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Brain Research 1054

Research Report

Localization of orexin-A-immunoreactive fibers in the

mesencephalic trigeminal nucleus of the rat

Irina I. Stoyanova*, Nikolai E. Lazarov

Department of Anatomy, Faculty of Medicine, Thracian University, P. O. Box 1025, BG-6010 Stara Zagora, Bulgaria

Accepted 25 June 2005

Available online 28 July 2005

Abstract

Orexin A is a neuropeptide located exclusively in neurons in the hypothalamic nuclei involved in the central regulation of many brain

functions, related to motor activity and state-dependent processes. Orexins modulate behavioral state via actions across multiple terminal

fields. In order to determine whether the mesencephalic trigeminal neurons may receive a direct hypothalamic orexinergic input, the

distribution of orexin A immunoreactivity was examined in the rat mesencephalic trigeminal nucleus (MTN), using orexin A

immunohistochemistry. Orexin-A-immunostained nerve fibers and terminals were found in a close apposition to the perikarya of primary

afferent neurons in the MTN with a marked rostrocaudal gradient in their density. In the caudal pontine MTN, only scattered orexin-A-

immunoreactive fibers were found, while more rostrally in the pons, and in the midbrain–pontine junction part of the nucleus, orexin-A-

immunopositive varicosities were relatively more abundant, located in close proximity to or often surrounding the neuronal profiles. At the

level of the inferior or superior colliculi, a large number of orexin-A-containing neuronal processes and terminal arborizations were observed

traveling toward and contacting mesencephalic trigeminal neurons, some of which were multipolar. The results of this study show that MTN

neurons receive orexin A hypothalamic innervation with a somatotopic arrangement of the projections in the nucleus. The central orexinergic

system may exert direct influence upon jaw movements at the level of the MTN and thus to participate in the control of feeding behavior.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Neuropeptides and behavior

Keywords: Orexin A; Immunohistochemistry; Feeding behavior; Mesencephalic trigeminal nucleus; Rat

1. Introduction

Orexin A and B, also known as hypocretins, are recently

discovered hypothalamic peptides, which are specific

ligands for two different receptors belonging to the G-

protein-coupled receptor family [29]. Orexins are located

exclusively in a relatively small population of neurons in the

posterior and lateral hypothalamic perifornical region and

the dorsomedial hypothalamic nucleus [7,25]. These are

areas which are classically established to be involved in the

central regulation of behavioral state and state-dependent

processes [6,30]. Orexin circuits have also been implicated

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.06.066

* Corresponding author. Fax: +359 42 302 19.

E-mail address: stoyanovai@yahoo.co.uk (I.I. Stoyanova).

in the facilitation of mastication and feeding behavior

[5,14,29]. In fact, orexinergic neurons give rise to an

extensive projection system, part of which innervate the

groups of neurons associated with the genesis of the

masticatory pattern and thus may contribute to the final

masticatory motor output (reviewed in [18]).

Despite the restricted location of orexinergic neuronal

somata, their fibers and terminals are widely distributed

throughout the brain. Orexinergic efferent projections are

found to reach the brainstem nuclei involved in the control of

feeding, including the trigeminal sensory and motor nuclei

[4,25,28,33]. A previous study has also reported that both the

orexin receptor-1 and -2 are expressed at protein and mRNA

levels in the mesencephalic trigeminal nucleus (MTN) of the

rat. On the other hand, orexin A has a high but equal affinity

(2005) 82 – 87

I.I. Stoyanova, N.E. Lazarov / Brain Research 1054 (2005) 82–87 83

for the two receptors, while orexin B has a 10-fold higher

affinity for orexin receptor-2 than for orexin receptor-1 [29].

However, except for a recent report on the innervation of the

rat MTN neurons from hypothalamic orexin B-immuno-

reactive fibers [32], little is known as to whether orexin-A-

containing neurons send descending projections to this

nucleus. More recently, Zhang et al. [33] revealed the

presence of orexin-A- and orexin-B-containing axonal

projections to the feline MTN, albeit with a different staining

density of the orexinergic fibers in the nucleus. Nevertheless,

data about the distribution and functioning of the orexinergic

system in the MTN in rats are far from complete.

MTN contains primary sensory neurons innervating jaw-

closing muscle spindles and mechanoreceptors associated

with the teeth [1,2,13]. In their turn, MTN neurons transfer

proprioceptive information to the muscle of mastication

through their monosynaptic connections with trigeminal

motoneurons for controlling jaw movements ([21], reviewed

in [16,17]). In addition, jaw-closing motoneurons receive

inputs from jaw-muscle spindle afferents through premoto-

neurons, located in the area surrounding the trigeminal

motor nucleus (see [23], and references therein). Further-

more, it has been reported that the proprioceptive afferent

feedback to trigeminal motoneurons is crucial for modulat-

ing and coordinating the activity of the masticatory muscles

during oral motor behaviors [20]. Hence, MTN neurons

constitute a neuronal circuit of the mastication control, an

important part of feeding behavior.

Therefore, the aim of the present study was to determine

whether the primary trigeminal afferent neurons in the rat

MTN may receive a direct input from hypothalamic orexin-

A-containing efferents. We examined the distribution of

orexin A reactivity by immunohistochemistry at the light

microscopic level with a subtype-specific antibody.

2. Materials and methods

2.1. Animals and tissue preparation

Eight adult male Sprague–Dawley rats (250–350 g b.w.)

were used in this study. All housing facilities and procedures

used were supervised and approved by the Animal Care and

Use Committee of the Thracian University and were

consonant with the guidelines established by the NIH. The

animals were deeply anesthetized with Ketanest (50 mg/kg

i.p., Pfizer, New York, USA) and transcardially perfused,

first with 100 ml heparinized cold 0.9% NaCl (1 U

heparin/ml saline) and followed by 500 ml 4% paraformal-

dehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4).

After perfusion, the brains were extracted, blocked, and

postfixed in the same fixative solution for 5–6 h at 4 -C, andthen cryoprotected in 20% sucrose in PBS overnight at 4 -C.The brains were embedded in TissueTek OCT compound

(Miles Inc., Elkhart, NI, USA), frozen, and 20 Am thick

sections were cut in a cryostat at �20 -C. The sections were

separated into five series, according to the method proposed

by Guillery and Herrup [10]. After rinsing in 0.1 M PBS,

each complete series of one-in-five frontal sections, stretch-

ing through the entire rostrocaudal dimension of the MTN,

were processed for orexin-A immunohistochemistry.

2.2. Immunohistochemistry

The immunohistochemical staining procedure was per-

formed on free-floating sections according to the ABC

(avidin–biotin–horseradish peroxidase) method [11]. Briefly,

specimens were treated with hydrogen peroxide (0.3% in

absolute methanol; 30 min) to inactivate endogenous

peroxidase, and the background was blocked with 5% normal

goat serum. The primary antibody, polyclonal rabbit anti-

orexin A (Oncogene, Cambridge, MA, USA, Cat.# PC345),

was applied for 24 h at room temperature. It was diluted

1:1000 in a solution that blocks non-specific antibody

binding and promotes penetration of the antibody into the

tissue, containing 1 ml 0.05% of the preservative thimerosal

(Fluka, Buchs, Switzerland), 1 ml 0.1% bovine serum

albumin, 1 ml 10% normal goat serum, 1 ml 0.01% sodium

azide, and 6 ml 0.1 M PBS. After rinsing in PBS, sections

were incubated with the secondary antibody, biotinylated

goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA,

USA), and diluted 1:160 in PBS containing 1% normal goat

serum and 0.1% Triton X-100, for 6 h at room temperature.

After washing the sections, the ABC complex (Vector, 6.25

Al/ml of each compound in PBS) was applied. Following

rinsing, peroxidase activity was visualized using 2.4% SG

substrate kit for peroxidase (Vector) in PBS for 5 min at room

temperature. To reveal the precise location of MTN neurons

and labeled fibers in the brainstem, we counterstained them

with 0.5% Neutral Red (Sigma, St. Louis, MO, USA).

Finally, the sections were dehydrated in a graded series of

alcohols, cleared in xylene, and coverslipped with Entellan

(Merck, Darmstadt, Germany).

Negative controls included sections that were incubated

in the absence of the primary antibody or in the presence of

non-immune normal serum in the same dilution as the

primary antibody, as well as antigen–antibody preabsorp-

tion experiments with the native antigen orexin A (10�6 M,

Oncogene, Cambridge, MA, USA, Cat.# PC345-100 UG) at

4 -C for 24 h.

In this study, the presence of orexin-A-immunoreactive

fibers and their distributional pattern in the MTN was

determined semi quantitatively from very sparse (T), sparse(+), and moderate (++) to dense (+++). The relative

expression levels were subjectively assessed by visual

comparison, and the results of the analysis are summarized

in Table 1.

2.3. Data analysis and photomicrograph production

After immunostaining, the sections were photographed

with AxioCam MRC digital camera linked to a Zeiss

Fig. 1. Section from the caudal pontine MTN. Most MTN neuronal

perikarya are located here. The orexin-A-labeled fibers (arrowheads) are

sparse and less pronouncedly stained, and some of them contact MTN

somata. This part of the nucleus is outlined by a dense network of orexin-A-

containing axonal varicosities (arrows) extending to the adjacent locus

coeruleus (LC). Scale bar = 50 Am.

Table 1

Summary of distributional patterns of orexin-A-immunoreactive nerve

fibers and terminals in the MTN of the rat

MTN region Fiber density

Caudal pole T

Rostral pons +

Mesencephalic–pontine junction ++

Midbrain portion +++

(T) very sparse; (+) = sparse; (++) = moderate; (+++) = dense.

I.I. Stoyanova, N.E. Lazarov / Brain Research 1054 (2005) 82–8784

Axioplan 2 research microscope. For the assessment of

orexin A expression in the MTN, sections from �6.72 to

�10.04 mm (from Bregma) were selected according to

Coggeshall and Lekan [3], and images were generated

through 5�, 10�, 40�, and 100� objectives. All digital

images were matched for brightness and contrast in Adobe

Photoshop 7.0 software.

Cell counts were performed at various rostrocaudal

locations within the MTN. The average perikaryal size of

the neurons was determined for each MTN, and cell bodies

were subsequently categorized as small (up to 30 Am in

diameter), medium (30–50 Am), or large (>50 Am), based

on a measure always taken through the section of cell that

contained a nucleus.

Fig. 2. (a) Photomicrograph of a rat MTN at the midbrain–pontine junction

where a large and distinct group of medium-to-large sized pseudounipolar

neuronal somata is located. Thick orexin-A-immunostained neuronal

processes are observed (arrows). Note that the axonal varicosities and

bouton-like dots are in the vicinity of the perikarya of MTN neurons. (b)

The same section at a higher magnification. Relatively more numerous

orexin-A-immunopositive varicosities (arrows) are detected in this part of

the nucleus, and these are in direct contact with MTN neurons. Scale bar in

panel (a) is 50 Am; scale bar in panel (b) is 25 Am.

3. Results

3.1. Specificity

No immunoreactivity for orexin A was detected in the

tissues when normal serum or antiserum absorbed with

orexin A antigen was used (not shown). Neuronal structures

were considered to be orexin-A-immunopositive when their

staining was clearly stronger than that in the background.

The immunoreactive fibers and terminals were readily

discernible at the light microscopic level by the presence

of a dark-gray immunoreactive product.

3.2. Distribution of orexin A immunoreactivity in the MTN

Orexin-A-containing nerve fibers were not uniformly

distributed in the MTN, and a marked rostrocaudal gradient

in the density of orexin A staining was observed. Orexin-A-

immunopositive dot-like structures, presumably nerve ter-

minals, were scattered from the rostral to the caudal part of

the MTN and apposed upon the perikarya of large

pseudounipolar neurons. In the caudal pontine MTN, where

most MTN neuronal perikarya were located, orexin-A-

labeled fibers were fewer in number and less pronouncedly

stained. This part of the nucleus was delineated by dense

networks of orexin-A-containing axonal varicosities, which

in turn extend toward the neighboring locus coeruleus and

parabrachial nuclei (Fig. 1). More rostrally in the pons and

in the midbrain–pontine junction part of the nucleus, where

a large and distinct group of medium-to-large in size

pseudounipolar neuronal somata was observed, orexin-A-

immunopositive varicosities were relatively more numerous

(Figs. 2a, b). Nerve fibers and their terminals exhibiting

orexin A immunoreactivity were seen in close proximity to

(Fig. 3) and/or to be entirely surrounding the neuronal

profiles. At the level of the inferior or superior colliculi, a

comparatively large number of orexin-A-containing fibers

and terminal arborizations were found in direct apposition to

or coursing over the surface of MTN neurons of all sizes

(Fig. 4). At that level, some multipolar neuronal somata

Fig. 5. A cluster of two multipolar (arrows) and several small pseudou-

nipolar MTN neurons in the rostral portion of the nucleus. Numerous

orexin-A-immunoreactive varicosities are observed in the neuropil in-

between. Scale bar = 50 Am.

Fig. 3. Orexin A immunohistochemistry at the level of the inferior colliculi.

Numerous perineuronal orexin-A-immunoreactive varicose fibers (arrows)

and terminal boutons are seen in close proximity to the MTN neuronal

profiles. Scale bar = 25 Am.

I.I. Stoyanova, N.E. Lazarov / Brain Research 1054 (2005) 82–87 85

were observed, and orexin-A-immunoreactive axonal pro-

cesses terminated on them (Fig. 5).

4. Discussion

It is well known that the orexinergic neuronal perikarya,

located in the hypothalamic nuclei, give rise to an extensive

projection system, parts of which innervate multiple regions

associated with the regulation of behavioral state, and

especially feeding behavior [6,30]. Our results demonstrate

for the first time that the MTN of the rat receives a direct

innervation by orexin-A-immunoreactive fibers. In addition,

we found very close contacts between the MTN neuronal

perikarya and orexin-A-containing axonal terminals. Taken

together, with the previously reported orexin B innervation

of rat MTN [32], the present data suggest that processing of

Fig. 4. High-resolution micrograph of a section taken through the

mesencephalic part of the nucleus. A large number of orexin-A-containing

neuronal processes and terminals are found in close apposition to large and

small MTN cell bodies. Note the two orexin-A-labeled boutons (arrows)

terminating on a medium-sized neuron. Scale bar = 25 Am.

orofacial proprioceptive information in rats is mediated by

the orexinergic input through the two orexin receptor-1 and

-2 located on MTN neurons [9]. However, in contrast to the

extensive distribution of orexin B-immunoreactive fibers

and terminal fields mostly in the middle and caudal portions

of the rat MTN [32], we now demonstrate that orexin-A-

containing axonal projections are predominantly found in

the more rostral levels of the nucleus, which contains the

somata of spindle afferents.

A striking feature of the MTN neurons is the differential

topographic arrangement of their cell bodies supplying the

jaw-closing muscles and periodontal tissue. In fact, masti-

catory muscle afferent neurons are more evenly dispersed

throughout the entire rostrocaudal length of the nucleus

without any somatotopic arrangement, while periodontal

receptor afferent MTN neurons are mainly concentrated in

its caudal part [8,26]. In this respect, our findings of closely

apposed orexin-A-containing fibers and terminals across the

whole extent of the MTN, like in muscle spindle afferents,

correspond well to the conclusions of Peever et al. [27] that

orexins are mainly related with the function of the

masticatory muscles, and orexinergic fibers may increase

their motor activity. Indeed, recent evidence has shown that

a central injection of orexin A, which stimulates both the

orexin receptors with high activity, potently enhances

locomotor activity, as well as a short-lasting increase in

feeding [31].

Unlike other primary sensory neurons, the MTN afferents

possess synaptic contacts on their somata [16] and function

as proprioceptive feedback to control masticatory as well as

other types of oral motor behavior [22,23]. Furthermore, the

circuitry reported by Luo and Dessem [19] presents an

interaction between some MTN neurons and thus offers the

possibility that they may act as premotor neurons capable of

integrating sensory information prior to reaching the

trigeminal motor nucleus. The close apposition of orexin-

A-labeled hypothalamic axonal terminals to the MTN

I.I. Stoyanova, N.E. Lazarov / Brain Research 1054 (2005) 82–8786

neurons, observed in the present study, can be regarded as

synaptic contacts. Our results, along with other published

data [27], also suggest that orexin A input may act on

presynaptic terminals within the MTN to modulate the

neurotransmitter (glutamate) release. Since the excitatory

synaptic transmission between MTN afferents and trigemi-

nal jaw-closing muscles is important for the genesis of

feeding rhythms [18,27], orexin signals from the hypotha-

lamus could indirectly cause motoneuronal excitation and

an increased masseter muscle activity. In other words, it can

be inferred that hypothalamic orexinergic nerves may be

involved in feeding responses via sensory and motor

innervation of neurons controlling the masticatory process.

This indicates a direct hypothalamic control on sensory

afferents related to feeding behavior. Such a viewpoint has

already been reported for orexin B-containing hypothala-

mic projections to the MTN and the trigeminal motor

nucleus [32].

Previous studies demonstrate that neurons projecting to

the MTN are located in the posterior and lateral hypothala-

mus, regions implicated in feeding behavior [15]. Moreover,

MTN neurons are largely innervated by histamine- [12] and

adenosine-deaminase-containing hypothalamic projections

[24]. Our present observations provide evidence that, in

addition to these inputs, MTN neurons are under the

influence of yet another descending neurotransmitter sys-

tem, namely the orexinergic neuron system.

In conclusion, this study shows that the MTN neurons

receive orexin-A-ergic hypothalamic input with somatotopic

arrangements of the axonal projections in the nucleus. In

addition, our findings suggest that the central orexinergic

system may influence both the masticatory and propriocep-

tive process at the level of the MTN and thus participate in

the automatic control of feeding behavior.

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